A Remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules

Article abstract of DOI:10.1021/ja104591m

Prostate specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase overexpressed in many forms of prostate cancer. Our laboratory has recently disclosed a class of small molecules, called ARM-Ps (antibody-recruiting molecule targeting prostate cancer) that are capable of enhancing antibody-mediated immune recognition of prostate cancer cells. Interestingly, during the course of these studies, we found ARM-Ps to exhibit extraordinarily high potencies toward PSMA, compared to previously reported inhibitors. Here, we report in-depth biochemical, crystallographic, and computational investigations which elucidate the origin of the observed affinity enhancement. These studies reveal a previously unreported arene-binding site on PSMA, which we believe participates in an aromatic stacking interaction with ARMs. Although this site is composed of only a few amino acid residues, it drastically enhances small molecule binding affinity. These results provide critical insights into the design of PSMA-targeted small molecules for prostate cancer diagnosis and treatment; more broadly, the presence of similar arene-binding sites throughout the proteome could prove widely enabling in the optimization of small molecule-protein interactions.

Full text of DOI:10.1021/ja104591m

Published on Web 08/20/2010
A Remote Arene-Binding Site on Prostate Specific Membrane
Antigen Revealed by Antibody-Recruiting Small Molecules
Andrew X. Zhang,† Ryan P. Murelli,† Cyril Barinka,‡ Julien Michel,†
Alexandra Cocleaza,† William L. Jorgensen,† Jacek Lubkowski,§ and
David A. Spiegel*,†,|
Department of Chemistry, Yale UniVersity, 225 Prospect Street, P.O. Box 208107, New HaVen,
Connecticut 06510-8107, Laboratory of Structural Biology, Institute of Biotechnology AS CR,
V.V.i., 14200 Prague 4, Czech Republic, Macromolecular Crystallography Laboratory, 539
Boyles Street, National Cancer Institute at Frederick, Frederick, Maryland 21702, and
Department of Pharmacology, Yale UniVersity School of Medicine, 333 Cedar Street,
New HaVen, Connecticut 06520
Received May 26, 2010; E-mail: david.spiegel@yale.edu
Abstract: Prostate specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase
overexpressed in many forms of prostate cancer. Our laboratory has recently disclosed a class of small
molecules, called ARM-Ps (antibody-recruiting molecule targeting prostate cancer) that are capable of
enhancing antibody-mediated immune recognition of prostate cancer cells. Interestingly, during the course
of these studies, we found ARM-Ps to exhibit extraordinarily high potencies toward PSMA, compared to
previously reported inhibitors. Here, we report in-depth biochemical, crystallographic, and computational
investigations which elucidate the origin of the observed affinity enhancement. These studies reveal a
previously unreported arene-binding site on PSMA, which we believe participates in an aromatic stacking
interaction with ARMs. Although this site is composed of only a few amino acid residues, it drastically
enhances small molecule binding affinity. These results provide critical insights into the design of PSMA-
targeted small molecules for prostate cancer diagnosis and treatment; more broadly, the presence of similar
arene-binding sites throughout the proteome could prove widely enabling in the optimization of small
molecule-protein interactions.
1. Introduction
possesses glutamate carboxypeptidase activity, and numerous
studies have focused on the identification of small molecules
Prostate cancer is a major public health threat against which
currently available therapeutic strategies are often ineffective.1,2
Our laboratory has recently disclosed a class of small molecules,
called ARM-Ps (antibody-recruiting molecules targeting prostate
cancer) that are capable of enhancing antibody-mediated im-
mune recognition of prostate cancer cells.3,4 ARM-Ps ac-
complish this by binding simultaneously to prostate-specific
membrane antigen (PSMA),5 a membrane-bound glycoprotein
that is overexpressed on prostate cancer cells, and to antidini-
trophenyl (anti-DNP) antibodies, which are present endog-
enously in the human bloodstream (Figure 1A and B).6 PSMA,
which is also known as glutamate carboxypeptidase II (GCPII),
is a well-studied molecular signature of prostate cancer cells
and has been exploited as a target in both therapeutic and
diagnostic strategies for patients with prostate cancer.7-11 PSMA
(4) For lead examples of other bifunctional materials that redirect
antibodies to target cells, see: (a) Parker, C. G.; Domaoal, R. A.;
Anderson, K. S.; Spiegel, D. A. J. Am. Chem. Soc. 2009, 131, 16393–
16394. (b) Shokat, K. M.; Schultz, P. G. J. Am. Chem. Soc. 1991,
113, 1861–1862. (c) Naicker, K. P.; Li, H.; Heredia, A.; Song, H.;
Wang, L. Org. Biomol. Chem. 2004, 2, 660–664. (d) Li, J.; Zacharek,
S.; Chen, X.; Wang, J. Q.; Zhang, W.; Janczuk, A.; Wang, P. G.
Bioorg. Med. Chem. 1999, 7, 1549–1558. (e) Perdomo, M. F.; Levi,
M.; Saellberg, M.; Vahlne, A. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
12515–12520. (f) Bertozzi, C. R.; Bednarski, M. D. J. Am. Chem. Soc.
1992, 114, 5543–5546. (g) Bertozzi, C. R.; Bednarski, M. D. J. Am. Chem.
Soc. 1992, 114, 2242–2245. (h) Krishnamurthy, V.; Quinton, L.; Estroff,
L.; Metallo, S.; Isaacs, J.; Mizgerd, J.; Whitesides, G. Biomaterials 2006,
27, 3663–3674. (i) Metallo, S. J.; Kane, R. S.; Holmlin, R. E.; Whitesides,
G. M. J. Am. Chem. Soc. 2003, 125, 4534–4540. (j) Carlson, C.; Mowery,
P.; Owen, R.; Dykhuizen, E. C.; Kiessling, L. ACS Chem. Biol. 2007,
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Fasella, E.; Kiessling, L. L. ChemBioChem 2007, 8, 68–82. (l) Lu,
Y.; You, F.; Vlahov, I.; Westrick, E.; Fan, M.; Low, P. S.; Leamon,
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4378–4383. (o) Rader, C.; Sinha, S. C.; Popkov, M.; Lerner, R. A.;
Barbas, C. F., III. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 5396–
5400. (p) Guo, F.; Das, S.; Mueller, B. M.; Barbas, C. F., III; Lerner,
R. A.; Sinha, S. C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 11009–
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† Yale University.
‡ Institute of Biotechnology AS CR.
§ National Cancer Institute at Frederick.
| Yale University School of Medicine.
(1) American Cancer Society, “Cancer Facts and Figures 2010”, 2010.
Available at http://www.cancer.org/Research/CancerFactsFigures/
CancerFactsFigures/cancer-facts-and-figures-2010 (accessed July 7, 2010).
(2) Olson, W. C.; Heston, W. D. W.; Rajasekaran, A. K. ReV. Recent
Clin. Trials 2007, 2, 182–190.
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D. A. J. Am. Chem. Soc. 2009, 17090–17092.
9
10.1021/ja104591m  2010 American Chemical Society
J. AM. CHEM. SOC. 2010, 132, 12711–12716 12711

A R T I C L E S
Zhang et al.
Table 1. Linker Length Dependence on PSMA Inhibitory Potency
Compound
X
n
IC50 (nM)a
Ki (nM)b
1, ARM-P0
2, ARM-P1
3, ARM-P2
4, ARM-P4
5, ARM-P6
6, ARM-P8
7, ARM-P12
8, MeO-P0
9, MeO-P2
10, MeO-P4
11, MeO-P8
12, MeO-P12
DNP
DNP
DNP
DNP
DNP
DNP
DNP
OMe
OMe
OMe
OMe
OMe
0
1
2
4
6
8
12
0
2
4
1.76 ( 0.41
1.05 ( 0.11
0.54 ( 0.18
0.46 ( 0.18
2.29 ( 0.60
3.29 ( 1.14
37.3 ( 16.2
30.5 ( 12.1
40.8 ( 9.4
0.078 ( 0.018
0.047 ( 0.005
0.024 ( 0.008
0.020 ( 0.008
0.101 ( 0.027
0.145 ( 0.050
1.65 ( 0.72
Figure 1. Structure and function of ARM-Ps (antibody-recruiting molecules
targeting prostate cancer). (A) ARM-Ps recruit anti-DNP antibodies to
PSMA-expressing prostate cancer cells and, thereby, bring about immune-
mediated cytotoxicity. (B) ARM-Ps are bifunctional and consist of an
antibody-binding terminus (ABT), a linker region, and a cell-binding
terminus (CBT).
1.35 ( 0.54
1.81 ( 0.42
30.2 ( 14.4
131.0 ( 57.6
165.2 ( 58.9
1.34 ( 0.64
8
12
5.79 ( 2.54
7.30 ( 2.60
capable of inhibiting this enzyme.12-17 ARM-Ps belong to a
class of glutamate urea compounds capable of inhibiting PSMA
with high potency.14
a IC50 values represent the mean of triplicate experiments. b Ki values
were calculated from IC50 and Km values using the Cheng-Prusoff
equation as described in the Supporting Information.
During the course of developing ARM-Ps, we observed that
bifunctional DNP-containing conjugates were strikingly more
potent than the parent glutamate urea compounds from which
they were derived. Furthermore, we also noted that potency
increases were correlated to the length of the linker regions
connecting the two poles of the molecule. Here we provide a
molecular basis for these findings, which involves the disclosure
of a preViously unreported arene-binding site on PSMA. These
conclusions are supported by extensive biochemical, crystal-
lographic, and computational studies.
1-12). These compounds consist of glutamate ureas linked to
DNP or methoxy groups by oxyethylene moieties of varying
lengths. They are named ARM-Px and MeO-Px, respectively,
wherein “x” corresponds to the number of oxyethylene units in
the linker. Evaluation of these compounds for their ability to
inhibit PSMA activity proved quite revealing.3 In all cases,
ARM-P derivatives were found to possess Ki values lower in
magnitude than their counterparts lacking DNP (compounds 1-7
versus 8-12). In some cases, the affinity difference was up to
2 orders of magnitude (compound 3 versus 9). This result
indicated to us that perhaps the DNP function itself might be
playing a role in binding PSMA. Such a hydrophobic interaction
involving an aromatic ring and PSMA was not completely
unexpected given the proximity of the glutamate-urea binding
site to a known hydrophobic pocket in PSMA.18,19 Indeed,
inhibitors containing hydrophobic functionality distal to the
glutamic acid moiety have exhibited high potency against
PSMA.13,20,21
A model involving binding of the DNP moiety to the
hydrophobic pocket adjacent to the S1 site did not explain the
decrease in affinity between ARM-P2 and derivatives with
shorter oxyethylene linkers (i.e., ARM-P0 and ARM-P1).
Indeed, one would expect ARM-P0 and ARM-P1 to exhibit
enhanced potency versus ARM-P2 because of the close proxim-
ity of the accessory hydrophobic pocket to the P1′ glutamate
binding cavity. The opposite trend suggested perhaps the
presence of an alternative hydrophobic binding site, situated at
a substantial distance away from this cavity. This hypothesis is
2. Results and Discussion
2.1. Dependence of Linker Length on Binding Affinity. To
evaluate in detail the effect of linker length on PSMA binding
affinity, we prepared various derivatives of ARM-P (Table 1,
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Antibody-Recruiting Molecules Targeting Prostate Cancer
A R T I C L E S
Table 2. Dependence of Ki on Substituents and Electronics of
Aromatic Ring
Compound
X
ELUMO (eV)a
IC50 (nM)b
Ki (nM)c
3, ARM-P2 DNP
-1.44
-0.92
-0.91
0.50
0.43
N/A
0.54 ( 0.18
1.78 ( 0.15
1.36 ( 0.18
16.6 ( 6.3
0.024 ( 0.008
0.078 ( 0.007
0.060 ( 0.008
0.73 ( 0.28
0.97 ( 0.44
15.1 ( 7.5
13
14
15
16
17
o-NO2-Ph
p-NO2-Ph
Ph
p-MeO-Ph
Cyclohexyl
21.9 ( 9.9
342.3 ( 170.5
Figure 2. Correlation between Ki values and ELUMO of aromatic ring
component of ARM-P analogue. Measured Ki values (mean of triplicate
experiments ( standard deviation) are plotted versus PDDG/PM3 LUMO
energies calculated using the BOSS software package.
a ELUMO energies of arene rings (X-NHMe) were calculated using the
PDDG/PM3 method implemented in the BOSS software package and
reported in electron-volts as detailed in the Supporting Information.
b IC50 values represent the mean of triplicate experiments. c Ki values
were calculated using IC50 and Km values via the Cheng-Prusoff
equation as outlined in the Supporting Information. A Km value of 925
nM was used in these calculations.
an additional order of magnitude less potent than mononitrated
derivatives (Ki ) 730 and 970 pM, respectively), and the
cyclohexyl-substituted derivative (17) proved yet another order
of magnitude worse than the least potent aryl compounds (Ki
) 15.1 nM). This may result from the enhanced steric bulk of
the cyclohexyl substituent versus planar arenes.
To examine the relationship between arene electronics effects
and PSMA inhibitory potency, we performed semiempirical
calculations of arene LUMO energies using the PDDG/PM3
method implemented in the BOSS software package.27,28 An
excellent correlation was observed between the LUMO energies
of aromatic substituents and experimentally determined Ki values
(Figure 2). Electron-poor aromatic rings are expected to experience
strong π-stacking interactions with electron-rich arenes,29,30 sug-
gesting that such interactions may be dominant in dictating binding
affinity in this system. These results are strongly indicative of
multisite binding in the ARM-P series and led us to test this
hypothesis further using X-ray crystallography.
supported by observations in related systems in which bifunc-
tional ligands bind proteins at two remote sites. In such systems,
an ideal linker length between binding poles is required for
maximum affinity; shorter linkers prevent formation of optimal
interactions in both binding sites, while longer linkers lead to a
relatively high entropic penalty upon bivalent binding without
any compensatory enthalpic benefit.22-25
Notably, compounds within the MeO-P series (8-12) con-
taining relatively short linkers all bind PSMA with comparable
affinity. The presence of linkers consisting of 8 oxyethylene
groups or longer appears to inhibit compound binding, perhaps
because bulky PEG chains prevent access of the glutamate urea
moiety to its binding site on PSMA.26 The increased sensitivity
of ARM-P derivatives to changes in linker length as compared
to MeO-P compounds suggests that factors other than simple
steric bulk are operating for the ARM-Ps.
2.3. Crystallographic Studies.
2.2. Structure-Activity Studies: Effect of Varying Aromatic
Groups on Binding Affinity. To test further our model for
bidentate binding, we set out to probe the impact of the phenyl
ring substituent on inhibitor potency. We therefore synthesized
analogues of ARM-P2 replacing the DNP moiety with a range
of electronically distinct aromatic species (3, 13-17, Table 2).
As shown, these analogues included nitrophenyl (ortho and para
to the linker), p-methoxyphenyl, phenyl, and cyclohexyl deriva-
tives. Consistent with the hypothesis that the DNP moiety
occupies a novel binding site, profound changes in affinity were
observed in this series. Interestingly, the parent dinitrophenyl-
containing compound (ARM-P2) possessed the highest potency
of all the analogues tested (Ki ) 24 pM), and removal of nitro
groups led to 3-fold decreases in affinity in the p-nitrophenyl
(13) and o-nitrophenyl (14) analogues (Ki ) 60 and 78 pM,
respectively). The similarities between these analogues suggest
that inhibitor potency is dictated by electronic rather than steric
effects. Phenyl (15) and methoxyphenyl (16) analogues proved
2.3.1. Initial Refinement and Analysis. Crystal structures were
determined for PSMA in complex with ARM-P ligands contain-
ing 2, 4, and 8 oxyethylene units in the linker region (3, 4, and
6) and with MeO-P4 (10), which lacks the DNP moiety, and
were refined at a resolution of 1.69, 1.59, 1.59, and 1.78 Å,
respectively. Individual compounds were fit into the positive
peaks on the difference Fo-Fc electron density map in the final
stages of refinement. For all four inhibitors, clear interpretable
densities were observed for the C-terminal part encompassing
the P1′ glutarate, the urea linkage, the lysine linker, and the
triazole ring. Although density corresponding to the DNP phenyl
ring is defined in all ARM-P complexes, density corresponding
to the nitro groups is absent suggesting that the DNP moiety is
present in at least two different conformations. Also, electron
density peaks corresponding to the poly(oxyethylene) linker
were absent from all complexes, consistent with a lack of
intermolecular contacts between this flexible element and the
protein.
(22) Contakes, S. M.; Juda, G. A.; Langley, D. B.; Halpern-Manners, N. W.;
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Freeman, H. C. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 13451–6.
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A R T I C L E S
Zhang et al.
Figure 3. Close-up of PSMA active site bound to bifunctional glutamate
urea inhibitors ARM-P2 (gold), ARM-P4 (gray), and ARM-P8 (blue).
Structures were superimposed on with corresponding (or equivalent) CR
atoms. Inhibitors are shown in stick representation, and protein residues
are shown as lines. Hydrogen bonding interactions are indicated by dashed
lines. The zinc ions and chloride ion in the active site are labeled as gray
and green dotted spheres, respectively, and water molecules are depicted
as red spheres. In both protein and inhibitor structures, carbon atoms are
colored as indicated above, and other atoms are colored red (oxygen) and
blue (nitrogen).
Figure 4. The PSMA/ARM-P2 complex reveals a previously unreported
arene-binding cleft. (A) Global view of PSMA with a close-up of arene-
binding site. Residues making up the arene-binding cleft are labeled in cyan.
The entrance lid (residues 542-548), which resides in an open conformation
in the ARM-P2 complex, is indicated as a red loop. Overlaid on this complex
is the entrance lid in its closed conformation (colored blue), which would
come into steric conflict with the linker region of the inhibitor. (B and C)
Close-up images of the urea binding sites in structures containing both open
and closed entrance loops (colored in blue). Residues forming the DNP
binding site (W541, R511, and R463) are colored red. In all panels, structural
data for PSMA with a closed entrance lid comes from the complex with
the small urea-based inhibitor DCIBzL (PDB ID: 3IWW).33
Models of ARM-P2, ARM-P4, and ARM-P8 in complex with
PSMA, built from the electron density map, are depicted in
Figure 3. Despite the attachment of large oxyethylene linkers,
the glutamate urea portions of all inhibitors interact with the
protein active site in a fashion reminiscent of previously reported
complexes with urea13,14,18 and phosphonate31 inhibitors, and
the substrate N-acetyl-aspartyl-glutamate (NAAG).32 In all
structures, positioning of the P1′ glutarate is enforced by
H-bonds (indicated as dashed lines) with Arg210, Asn257,
Tyr552, Lys699, Tyr700, and active-site water molecules, and
hydrophobic interactions with the side chains of Phe209 and
Leu428. The ureido nitrogen atoms serve as H-bond donors in
interactions with Glu424 and the Gly518 main chain carbonyl,
and the carbonyl oxygen makes contacts with the catalytic zinc
atom, Tyr552, and His553. The P1 R-carboxylate in all inhibitors
structurally overlaps with the equivalent groups of previously
reported complexes and is held in place by interactions with an
arginine-rich patch (Arg463, Arg534, Arg536) along with
H-bonding contacts to Asn519, the Ser517 main-chain carbonyl,
and water molecules.18,19
2.3.2. Discovery of an Arene-Binding Cleft. A key site of
interaction between PSMA and all ARM-P derivatives is the
triazole ring, which was observed to pack against the side chains
of Tyr552 and Tyr700 in all complexes (Figure 3). The steric
hindrance caused by the oxyethylene linker emanating from the
triazole ring prevents closure of the enzyme’s entrance lid
(amino acids Trp541-Gly548), as observed for PSMA complexes
with smaller ligands.19 A key consequence of the entrance lid’s
open conformation is the revelation of a previously unreported
binding cleft for the DNP ring (Figure 4).19
The zinc ions
in the active site are labeled as orange spheres, and the ARM-P2 carbons
are colored gold. The DCIBzL carbons in B and C are colored purple.
virtually parallel to both indole and guanidinium functionalities,
suggesting that simultaneous π-cation (DNP-Arg511) and
π-stacking (DNP-Trp541) interactions may both contribute to
inhibitor binding.34,35 Critically, the arene-binding region is only
revealed upon opening of the entrance lid (Figure 4B); closure
of the entrance lid, as in the overlaid complex between PSMA
and the small urea-based inhibitor DCIBzL,33 would lead to
significant steric overlap with the triazole moiety as well as
closure of the arene-binding site (Figure 4C). Thus, the protein
is capable of adopting two separate conformations, each suited
to accommodate high-affinity binding interactions with distinct
classes of glutamate-urea inhibitors.
A key structural feature was observed in the MeO-P4 complex
(Figure 5). Here, unlike in the ARM-P complexes, Trp541 exists
in two distinct conformations. The nonstacking conformation
is rotated approximately 4 Å from what is seen in ARM-P
complexes and blocks the arene-binding groove. The confor-
mational flexibility exhibited by Trp541 in the PSMA/MeO-P4
complex suggests that when present, the dinitroarene stabilizes
the side chain indole moiety via π-stacking, as implied by the
ARM-P structures depicted above. Taken together, these data
provide strong support for a model in which ARM-Ps bind
PSMA through interactions both at the enzyme active site and
at a newly reported arene-binding cleft.
The arene-binding region, formed from the indole group of
Trp541 and the guanidinium group of Arg511, holds the DNP
ring in close contact with these groups at distances of 3.1 and
3.9 Å, respectively. The bottom of the cleft is lined by the
Arg463 side chain. Positioning of the phenyl ring creates a plane
Notably, the complex between PSMA and MPE,36 a meth-
otrexate-derived phosphonate, was also shown to possess an
(31) Barinka, C.; Rovenska, M.; Mlcochova, P.; Hlouchova, K.; Plecha-
novova, A.; Majer, P.; Tsukamoto, T.; Slusher, B. S.; Konvalinka, J.;
Lubkowski, J. J. Med. Chem. 2007, 50, 3267–3273.
(34) Duffy, E. M.; Kowalczyk, P. J.; Jorgensen, W. L. J. Am. Chem. Soc.
1993, 115, 9271–9275.
(32) Klusa´k, V.; Barˇinka, C.; Plechanovova´, A.; Mlcˇochova´, P.; Konvalinka,
J.; Rulísˇek, L.; Lubkowski, J. Biochemistry 2009, 48, 4126–4138.
(33) Wang, H.; Byun, Y.; Barinka, C.; Pullambhatla, M.; Bhang, H.-e. C.;
Fox, J. J.; Lubkowski, J.; Mease, R. C.; Pomper, M. G. Bioorg. Med.
Chem. Lett. 2010, 20, 392–397.
(35) Gallivan, J. P.; Dougherty, D. A. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 9459–64.
(36) The abbreviation MPE is used to stand for 2-[(3-{4-[(2-amino-4-
hydroxy-pteridin-6-ylmethyl)-amino]-benzoylamino}-3-carboxy-pro-
pyl)-hydroxy-phosphinoylmethyl]-pentanedioic acid.
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Antibody-Recruiting Molecules Targeting Prostate Cancer
A R T I C L E S
Figure 5. Close-up view of the active site of PSMA bound to MeO-P4.
Hydrogen bonding interactions are indicated by dashed lines. The zinc ions
in the active site and adjacent chloride ion are labeled as gray and green
dotted spheres, respectively, and water molecules are depicted as red spheres.
In both protein and inhibitor structures, carbon atoms are colored in olive,
and other atoms are colored red (oxygen) and blue (nitrogen).
open entrance lid like the complexes disclosed herein.19 It was
concluded from the PSMA/MPE complex that the protein’s
ability to adopt an open conformation serves to enable its
binding to relatively large substrates, such as folyl-poly-γ-
glutamates. One might imagine that the revelation of an arene-
binding site upon opening of the entrance lid might serve to
enhance affinity for these arene-containing enzyme substrates.
Interestingly, however, the pendant pteroyl ring in the MTE
complex was not observed to interact with the PSMA arene-
binding cleft, perhaps due to its relatively short linkage to the
zinc-binding phosphonate region. The observations reported
herein suggest that perhaps larger natural poly-γ-glutamate
substrates are able to make use of the arene-binding site;
however further studies are necessary to test this possibility.
2.4. Molecular Dynamics (MD) Simulations. To clarify the
nature of the protein-ligand interactions in the ARM-P
complexes, explicit solvent molecular dynamics (MD) simula-
tions were carried out using crystallographic data for PSMA
complexes with ARM-P0, ARM-P2, ARM-P4, ARM-P8, and
MeO-P0. Each protein-ligand complex was modeled with the
OPLS-AA force field,37 embedded in a triclinic box of TIP3P
water molecules.38 Dynamics were simulated for 50 ns using
the Desmond software package.39
Figure 6. Selected snapshots from the MD simulations of PSMA/ARM-P
complexes. The ligands are represented in colored sticks; Arg463, Arg511,
and Trp541 are represented in orange sticks. Figure created with the program
VMD.40
These observations also explain the absence of electron density
corresponding to linker regions in all crystal structures.
By far the most stable intermolecular contact in the arene-
binding site in ARM-P-PSMA complexes is the stacking
interaction between DNP and Trp541. For all ARMs, the DNP
moieties participate in face-to-face interactions with Trp541 side
chain indole moieties for significant time periods throughout
MD simulations. Simulations of the ARM-P0-PSMA complex
revealed a remarkable level of flexibility in the triazole-alkyl
region, which enables π-stacking contacts in the arene-binding
site to remain intact even in the absence of an oxyethylene linker
(Figure 6d-f). When stacked with the Trp541 side chain indole,
the DNP ring is observed to rotate in-plane, supporting the
hypothesis that the lack of well-defined electron density
corresponding to nitro groups in crystal structures is due to the
presence of multiple arene conformations. However, in all
ARM-P complexes, the nitro groups in the DNP ring are
frequently observed pointing toward the Arg463 side chain
guanidinium group, suggesting possible hydrogen bonding or
electrostatic interactions between these groups. Furthermore,
although crystallographic data support a role for π-cation
interactions with Arg511 in the arene-binding site, this residue
is highly disordered in MD simulations and does not form long-
lived contacts with the ligand. This observation is consistent
with the data presented in Table 2 and Figure 2, which suggest
These simulations revealed a number of noteworthy features
(see Supporting Information for video files for all simulations).
Although the PSMA active site and glutamate urea moieties
are fairly rigid throughout the time scale of MD simulations,
distal protein-ligand interactions exhibit highly dynamic be-
havior. For example, the simulation of the MeO-P0-PSMA
complex revealed that the arene-binding site is unstable in the
absence of DNP; Trp541 tends to rotate toward Arg511, thus
obscuring the arene-binding site (Figure 6a-c). This observation
directly correlates with the disorder in Trp541 observed in the
MeO-P4-PSMA crystal structure (Figure 5). Furthermore, the
PEG moieties in all complexes are highly dynamic and do not
seem to form specific interactions with PSMA, suggesting that
these make minimal enthalpic contributions to binding affinity.
(37) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. J. Am. Chem. Soc.
1996, 118, 11225–11236.
(38) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.;
Klein, M. L. J. Chem. Phys. 1983, 79, 926–935.
(39) Bowers, K. J.; Chow, E.; Xu, H.; Dror, R. O.; Eastwood, M. P.;
Gregersen, B. A.; Klepeis, J. L.; Kolossvary, I.; Moraes, M. A.;
Sacerdoti, F. D.; Salmon, J. K.; Shan, Y.; Shaw, D. E. Proceedings
of the 2006 ACM/IEEE SC|06 Conference; Tampa, FL, 2006.
(40) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graph. 1996, 14, 33–
38.
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A R T I C L E S
Zhang et al.
that cation-π interactions play a relatively minor role versus
π-stacking interactions in stabilizing these systems.
either case, although structural data exist demonstrating the
unique propensity of nitroarenes to engage in π-stacking
interactions with aromatic amino acid side chains,46,47 the
proteomic prevalence of nitroarene-binding motifs has not been
systematically explored. The widespread existence of such
binding sites could enable facile optimization of small molecule
ligands for proteins identified through high-throughput screening
and could find ready utility in fragment-based approaches to
ligand design.48
Although underexplored, strategies that utilize small mol-
ecules to enhance recognition of pathogens by the human
immune system promise to leverage the strengths of both
antibody- and small-molecule-based therapeutic approaches. The
results reported herein suggest the possibility for improving such
technologies for treating prostate cancer. For example, ultrahigh-
affinity ARM-Ps could be constructed by exploiting the presence
of the arene-binding site in PSMA and converting the highly
flexible first-generation ARM-Ps into more rigid scaffolds. More
broadly, the high-level expression of PSMA (GCPII) on prostate
cancer cell surfaces and on tumor neovasculature,49 as well as
its putative role in the pathophysiology of schizophrenia,50 has
rendered it an extremely useful and popular target for inhibitor
design. The results presented herein therefore could substantially
impact the development of effective diagnostic and therapeutic
approaches for patients suffering from cancer and other diseases.
Notably, during the course of MD simulations for both ARM-
P2 (Figure 6g-i) and ARM-P8 (Figure 6m-o), the DNP ring
dissociates from the arene-binding cleft, whereas this interaction
remains intact in the ARM-P4 simulation (Figure 6j-l). Taken
together, these data suggest that the DNP-Trp541 interactions
are relatively weak. Interestingly, the contact with Trp541
reforms rapidly during the simulations of ARM-P2, but not
ARM-P8; this may reflect either a higher entropic penalty
associated with bivalent binding because of the longer linker
group or the tendency of the linker to occupy the arene-binding
site, thus preventing the DNP group’s return to Trp541.22-25
Additional studies to clarify the contribution of the linker group
to the binding thermodynamics of the ARM-P ligands would
be desirable. From a functional standpoint, the propensity of
ARM-P8 to disengage from the PSMA arene-binding site
enables it to form ternary complexes with prostate cancer cells
and antibodies, which is critical to its cytotoxic activity.3
However, this functionality comes at the expense of PSMA
binding affinity. Thus, the model reported herein suggests the
possibility of ultrahigh-affinity ARM-P analogues capable of
interacting simultaneously with the PSMA arene-binding site
and anti-DNP antibodies.
3. Conclusion
Acknowledgment. This work was funded by the National
Institutes of Health through the NIH Director’s New Innovator
Award Program (DP22OD002913 to D.A.S.), and through NIH
Grant GM32136 (to W.L.J.). C.B. acknowledges financial support
from the EMBO Installation Grant (#1978) and the Institutional
Research Support of the IBT (CEZ:AV0Z50520701). The use of
the Advanced Photon Source was supported by the U.S. Department
of Energy (W-31-109-Eng38). J.L. acknowledges financial support
from the National Cancer Institute, Center for Cancer Research.
This research was partially supported by the National Science
Foundation through Teragrid resources provided by the Texas
Advanced Computing Center under grant number TG-CHE090106
(to J.M.), and by a Marie Curie International Outgoing Fellowship
(J.M.) within the 7th European Community Framework Programme
(FP7-PEOPLE-2008-16704-1-IOF, 234796-PPIdesign).
Here we detail the discovery of an arene-binding site on
prostate-specific membrane antigen (PSMA), which gives rise
to unusually high affinity binding interactions with designed
bifunctional antibody-recruiting small molecules (ARMs). Our
conclusions are supported by extensive crystallographic, bio-
chemical, and computational data, which, taken together,
strongly suggest a model in which bidentate binding of ARM-
Ps to PSMA leads to substantial increases in inhibitor potency.
The serendipitous nature of the discovery reported herein along
with the relative simplicity of the PSMA arene-binding
siteswhich consists merely of three amino acids only one of
which (Trp541) is responsible for affinity enhancementssuggests
that low-affinity binding sites for arenes could be quite prevalent
among proteins. Along these lines, it is well-documented that a
large proportion of circulating immunoglobulin possess high-
affinity binding activity against nitroarene ligands,41 and between
1 and 10% of myeloma proteins bind nitrophenyl ligands.42 The
possibility that such binding sites arise from conserved folds
within immunoglobulin domains has been suggested;43 however,
this trend may also result from the unique immunogenicity of
nitroarenes,44,45 a property that has also been attributed to their
propensity to form hydrophobic contacts with proteins.44 In
Supporting Information Available: Detailed experimental
procedures and compound characterizations as well as videos
of simulations are provided. This material is available free of
charge via the Internet at http://pubs.acs.org.
JA104591M
(45) Gru¨newald, J.; Tsao, M.-L.; Perera, R.; Dong, L.; Niessen, F.; Wen,
B. G.; Kubitz, D. M.; Smider, V. V.; Ruf, W.; Nasoff, M.; Lerner,
R. A.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 11276–
80.
(41) Antibodies recognizing the 2,4-dinitrophenyl (DNP) epitope have been
estimated to constitute 1% of circulating IgM (approximately 10 µg/
mL in human serum) and 0.8% of circulating IgG (approximately 40-
120 µg/mL in human serum). See: (a) Karjalainen, K.; Makela, O.
Eur. J. Immunol. 1976, 6, 88–93. (b) Farah, F. S. Immunology 1973,
25, 217226). (c) Rowe, D. S.; Anderson, S. G.; Skegg, J. In
Immunoglobulins; Merler, E., Ed.; National Academy of Sciences
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(47) Nitroarene-protein complexes were downloaded from the RCSB
protein data bank and include the following PDB IDs: 1VID, 3BWM,
3BWY, 3GE5, 1RSM, 1W4O, 1NEN, 2ROY, 2B14, 2B15, 2B16,
2RAZ, 1D1A, 1D1B, 1D1C, 2BMQ, 1BAF, 2BFG, 1H2J, 4A3H,
1QI2, 1OAU, 1GW1, 1GVY, 1IDT, 1OO6, 1GVO, 1GVS, 1VYP,
1VYR, 1VYS, and 1GVR. Out of a total of 32 available complexes,
16 indicated π-stacking interactions between the nitroarene and an
aromatic amino acid side chain.
(42) Michaelides, M. C.; Eisen, H. N. J. Exp. Med. 1974, 140, 687–702.
(43) Parker, C. W.; Osterland, C. K. Biochemistry 1970, 9, 1074–82.
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M.-L.; Perera, R.; Kang, M.; Laffitte, B. A.; Azarian, S.; Ruf, W.;
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